Nephrology Dialysis Transplantation

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1 Nephrol Dial Transplant (1998) 13: Original Article Nephrology Dialysis Transplantation Bone markers in the diagnosis of low turnover osteodystrophy in haemodialysis patients Giorgio Coen, Paola Ballanti1, Ermanno Bonucci1, Santo Calabria, Micaela Centorrino, Valeria Fassino, Micaela Manni, Daniela Mantella, Sandro Mazzaferro, Ilaria Napoletano, Daniela Sardella and FrancoTaggi2 Chair of Nephrology, Pathophysiology and Hypertension Unit and 1Department of Experimental Medicine and Pathology, La Sapienza University and 2Ist Superiore di Sanita, Rome, Italy Abstract the other groups by humoral parameters, at the highest Background. Renal osteodystrophy includes a number value of accuracy, showed 100% sensitivity and 93.7% of low and high turnover bone histologic patterns specificity with a cut-off of 12.9 ng/ml for BALP; which require a bone biopsy for their full identification. 88.9% sensitivity and 93.7% specificity with a cut-off The role of intact PTH and several classical and more of 21.5 ng/ml for DPD, and 88.9% sensitivity and recent bone markers in the non-invasive diagnosis of 90.6% specificity with a cut-off of 79.7 pg/ml for intact renal bone disease in patients with CRF in HD requires PTH. The other markers had lower values. A standardized further definition since available published data are z-score approach for evaluation of all humoral limited. parameters was also carried out. Using all variables, a Methods. In addition to intact PTH, alkaline phosphatase correct classification of MO/HP and of LTO was (AP) and osteocalcin (BGP), bone alkaline possible in 93.8 and 88.9% of the cases, respectively. phosphatase isoenzyme (BALP), tartrate resistant acid Predictive power was 96.8 and 80%, respectively for phosphatase ( TRAP), C-terminal cross-linked peptide MO/HP and LTO. When the only variables used were of collagen type 1 ( ICTP) and deoxypyridinoline intact PTH and BALP, a correct classification of (DPD) were measured in the serum of 41 patients on MO/HP and LTO was possible in 90.6% and 88.9%, haemodialysis, subjected at the same time to transiliac respectively. Predictive value of MO/HP was 96.7% bone biopsy for histomorphometric, histodynamic and and for LTO 72.7%. Predictive values using PTH and aluminium histochemical examination. Histodynamic AP were 96.3% and 57.2%, respectively. evaluation following double tetracycline label, was Conclusion. Intact PTH and several relatively new carried out in 37 patients. The patients had no evidence bone markers are of certain value in the non-invasive of active cytolytic and cholestatic liver disease and a diagnosis of renal osteodystrophy. However some of history of very limited aluminium exposure. the humoral markers carry the same quality of informa- Results. The patients had differing degrees of hyper- tion and the use of intact PTH and BALP may be parathyroidism, with intact PTH ranging from normal adequate in the discrimination of bone histologic pat- to very elevated levels. Serum values of the markers terns. In cases exempt from liver disease, PTH and AP BGP, ICTP and DPD, normally excreted through the may be used as a less costly alternative. Bone biopsy kidneys, were on average very high. The correlation could be chiefly limited to cases with borderline humoral coefficients of the humoral parameters vs dynamic values and to all those with a suspected aluminium variables, such as BFR/BS, were high. The highest overload. values were: intact PTH 0.798, AP 0.900, BALP 0.891, ICTP The patients, grouped in low turnover Key words: renal osteodystrophy; adynamic bone osteodystrophy (LTO; 9), mixed osteodystrophy disease; bone biopsy; intact PTH; bone markers; osteo- ( MO; 9) and prevalent hyperparathyroidism ( HP; calcin; tartrate resistant acid phosphatase; deoxy- 23), showed significant difference in the levels of piridinoline; bone alkaline phosphatase isoenzyme; most humoral and static and dynamic parameters C-terminal telopeptide of collagen type I (ANOVA). Bone aluminium histochemistry was negative in all cases. Discrimination of LTO patients from Introduction Correspondence and offprint requests to: Giorgio Coen, MD, Pathophysiology and Hypertension Unit, Institute of 2nd Clinica Medica, Policlinico Umberto I, Viale del Policlinico, I Renal osteodystrophy is a metabolic bone disease Rome, Italy. occurring in dialysis and non-dialysis patients with 1998 European Renal Association European Dialysis and Transplant Association

2 Bone markers and renal osteodystrophy 2295 advanced chronic renal failure. Histopathologically it renal failure and who were treated with standard haemodia- ranges from high turnover disease typical of frank lysis for an average of 82.6±52.67 months. All patients were hyperparathyroidism, and mixed forms, a combination oligoanuric and ESRF was due to: chronic glomerulo- of hyperparathyroid and osteomalacia lesions and low nephritis [14], interstitial nephropathy [10], ADPKD [6 ], nephrosclerosis/ischaemic renal disease [7], diabetic nephroturnover bone disease. Renal osteodystrophies exhibitpathy [1], and unknown [3]. They had received mainly ing a low turnover pattern are mainly osteomalacia calcium salts as chelating agents. Sixteen patients, in addition and adynamic bone disease (ABD) [ 1]. Osteomalacia to phosphate chelating calcium salts, received for relatively in haemodialysis patients is viewed as a consequence short periods of time small doses of aluminium hydroxide of aluminium intoxication [2,3], while it is mainly (1 2 g daily) in case of transitory diminished control of attributed to metabolic acidosis in chronic renal serum phosphate concentrations. The other patients had failure patients not treated with haemodialysis [4,5]. discontinued aluminium hydroxide therapy at least 2 years Adynamic bone disease is a term which describes a before the study. At the time of the study, none of the histological pattern of renal osteodystrophy once conhad been on calcitriol oral medication, 0.25 mg daily, which patients were receiving vitamin D and only a minority [14] sidered to be an aluminium related bone disease and was discontinued at least 4 weeks before the study. The was later found to be unrelated to aluminium accumupatients did not receive corticosteroids, anticonvulsants, antilation in most cases [6]. This bone lesion is observed coagulants, or NSAIDs. None of the patients had evidence in the majority of low turnover osteodystrophies and of liver disease as evaluated by serum liver enzyme aminois also the most frequent bone histologic finding in transferases (AST, ALT) and gammaglutamyl transpeptidpatients on peritoneal dialysis and haemodialysis [1]. ase (GGT ). Histologic, histomorphometric, hystodynamic, and On a non dialysis day, a blood sample was collected for histochemical examinations are required to form a calcium, phosphate, total alkaline phosphatase (AP), bone complete diagnostic definition of the wide variety of alkaline phosphatase isoenzyme (BALP), osteocalcin (BGP), bone histological patterns found in chronic renal failtype I (ICTP), tartrate resistant acid phosphatase ( TRAP) intact PTH, 25OHD, C-terminal telopeptide of collagen 3 ure, with presumably different pathogenetic mechanand deoxypyridinoline (DPD). isms requiring different therapeutic approaches. After informed consent, the patients were also subjected However, in many cases, clinical and laboratory tech- to transiliac bone biopsy. Bone biopsies were performed in niques could be used to avoid this invasive procedure, all patients with a Bordier trocar, 5 mm internal diameter. which can only be performed in specialized centres. In 37 patients, the biopsy was taken following double tetra- Despite much progress in the development of biochem- cycline administration at a 2-week interval (demeclocycline ical markers of metabolic bone disease, diagnosis 300 mg p.o. T.I.D. for 2 days each course). Aluminium remains uncertain and bone biopsies are often required. histochemical staining and histological, histomorphometric, In the last years, beside the marked progress in the and histodynamic examinations were performed. Blood radioimmunometric assays of parathyroid hormone, samples for the determination of serum parameters were several markers of osteoblastic and osteoclastic deriva- drawn at the same time. Serum intact ipth was measured with a commercial tion have been proposed for the non invasive diagnosis ( Incstar, Stillwater, USA) immunoradiometric assay based of renal osteodystrophy [7]. PTH has been found to on a double antibody technique. Intra- and interassay varibe a relatively imprecise predictor of bone lesions in ations were 6.5 and 9.8%, respectively. Normal mean value renal osteodystrophy [8,9], and the other more classical is 20.8±7.43 pg/ml (range pg/ml ). Serum osteocalcin, markers, osteocalcin and total alkaline phosphatase or BGP, was measured by radioimmunoassay based on the are considered less valid than PTH [7]. However, other method of Price and Nishimoto [13]. The intra- and interas- bone markers have been identified with a potential of say variations were less than 5 and 8%, respectively. Normal improving the non-invasive diagnosis. Bone alkaline mean value 3.89±1.45 ng/ml. phosphatase isoenzyme as an indicator of osteoblastic Serum 25-hydroxycholecalciferol was measured with a activity [10] and tartrate resistant acid phosphatase, competitive protein-binding method, after purification on Sep-Pack C18 cartridges and extraction with acetonitrile. serum C-terminal telopeptide of collagen type I and Normal range ng/ml. An immunoradiometric assay pyridinolin crosslinks as indicators of the resorption with a sensitivity of 2 ng/ml was used to measure bone process, can be specifically measured to estimate bone alkaline phosphatase (BALP) ( Tandem-Ostase,Hybritech turnover and are potentially important for non- Europe, Belgium). Intra- and interassay variabilities were 4.2 invasive diagnosis of renal osteodystrophy [11,12]. and 7.2%, respectively. Normal values 11.8±4.3 ng/ml. The purpose of this study was to compare intact Tartrate resistant acid phosphatase ( TRAP) of serum was PTH and several of the classical and more recent evaluated by colorimetric assay by measuring the osteoclastic markers of bone disease in the non invasive evaluation derived isoenzyme activity which is not inhibited by sodium of the histologic pattern and the degree of bone tartrate. The substrate is a-naftil-phosphate. Normal values turnover in haemodialysis patients. are <6.5 mu/ml. The TRAP assay was performed on serum from only 26 (Figure 1) of the patients with bone biopsies evaluated for histodynamic parameters. C-terminal cross-linked peptide of collagen type I ( ICTP) Subjects and methods is a small peptide of collagen generated in the resorption process by osteoclasts and was determined with a radioimmunoassay The study was peformed on 41 haemodialysis patients, from from Orion Diagnostica (Oulu, Finland). The several haemodialysis units in Rome. They were 18 females rabbit antibody was directed against human ICTP obtained and 23 males (average age 51.29±12.4 years), with chronic after bacterial collagenase digestion of femoral-derived bone

3 2296 Fig. 1. Correlations among serum levels of bone markers and BFR/BS. collagen. Precipitation of the antigen-antibody complex was obtained with a second antibody/peg solution. Intra- and interassay coefficients of variation were 4.9% and 7.1%, respectively. Detection limit for the assay was 0.5 ng/ml. Normal values are within 1.8 and 5 ng/ml serum. Serum free deoxypyridinoline liberated in the osteoclastic G. Coen et al. resorption was detected by radioimmunoassay (Nichols Inst., CA, USA) using an antibody derived from sheep. Sensitivity of the assay is 0.24 nmol/l. Intra- and interassay variability were 4.3 and 5.4%, respectively. Normal values nmol/l. Serum total calcium was determined by atomic absorption spectrophotometry (Perkin Elmer, model 2380, Norwalk, CT, USA; normal values mg/dl ). Serum phosphate and alkaline phosphatase measurements were performed spectrophotometrically (DU-65 Beckman, Fullerton, USA) using molibdate or p-nitrophenyl-phosphate as respective substrates. The normal range for the adult population is mg/dl and mu/ml, respectively. Bone specimens were fixed in 4% paraformaldehyde in 0.1 M phosphate buffer (ph 7.2). They were then longitudinally halved, dehydrated in acetone, and embedded without decalcification, using the JB-4 glycol-metacrylate embedding mixture (Polysciences, Inc., Warrington, PA) as previously described [14]. Sections were cut on a Reichert-Jung Autocut microtome equipped with a tungsten carbide knife. Three 4 mm thick sections of the specimens, together with positive controls, were stained by the aluminium histochemical staining technique according to the method of Maloney et al. [15]. Alternate sections, 1 2 mm thick, were stained with azure II-methylene blue for histomorphometric measurement of structural and static variables. Alternate sections, ~5 mm thick, were also prepared unstained to be examined under UV light for histodynamic evaluation of tetracycline fluorescent labels. Histomorphometric and histodynamic measurements were obtained using an interactive colour videobased image analysis system ( IAS 2000, Delta Sistemi, Rome, Italy) with a personalized software developed for bone histomorphometry. The following variables were measured [16 ]: bone volume (BV/TV, %), per cent of whole trabecular bone volume occupied by calcified and uncalcified bone tissue; osteoid volume (OV/BV, %), per cent of bone volume consisting of osteoid; osteoid thickness (OTh, mm),thickness of osteoid seams; osteoid surface (OS/BS, %), per cent of trabecular surface covered by osteoid; osteoblast surface (ObS/BS, %), per cent of trabecular surface covered by osteoid lined by active osteoblasts; eroded surface ( ES/BS, %), per cent of trabecular surface consisting of Howship s lacunae; osteoclastic surface (OcS/BS, %), per cent of trabecular surface covered by osteoclasts and undergoing resorption; single labelled surface (sls/bs, %), per cent of trabecular surface single labeled with tetracycline; double labelled surface (dls/bs, %), per cent of trabecular surface double labeled with tetracycline; mineralizing surface (MS/OS, %), the extent of double labeled plus half the extent of single labeled, as per cent of osteoid surface; mineral apposition rate (MAR, mm/day), the distance between the midpoints of two consecutive labels, divided by the time interval between the midpoints of the two labeling periods; bone formation rate, (BFR/BS, mm3/mm2/day), the volumetric amount of new mineralized bone per unit of trabecular bone surface per day; adjusted appositional rate (Aj.AR, mm/day), the mineral apposition rate over the entire osteoid surface; and mineralization lag time (Mlt, days), the mean time interval between deposition of osteoid matrix and its mineralization. BV/TV was evaluated at objective magnification of 2.5 and static and dynamic variables were evaluated at objective magnification of 10 [17]. Normal values ( Table 1) for the histomorphometric [18] and histodynamic [19] parameters were obtained in our own histomorphometric laboratory. The classification of different types of renal osteodystrophy was made on the basis of morphological criteria [19]. The

4 Bone markers and renal osteodystrophy 2297 Table 1. Median and mean values (±SD) of clinical data, humoral variables and histomorphometric and histodynamic parameters Patients Normal valuesa Median Min Max Mean (SD) Age, years ±12.40 Sex, F/M 18/23 HD age, month ±52.67 Ca,mg/dl ± s P,mg/dl ± s 25 OHD, ng/ml ± PTH intact, pg/ml ± BGP (osteocalcin), ng/ml ± AP, U/l ± BALP, mg/l ± ±4.30 TRAP, U/l ± ICTP, ng/ml ± DPD (deoxypyridinoline), nmol/l ± BV/TV, % ± ±4.50 OV/BV, % ± ±1.08 O.Th, mm ± ±3.32 OS/BS, % ± ±6.88 ObS/BS, % ± ES/BS, % ± ±1.28 OcS/BS, % ± ±0.19 sls/bs, % ± ±2.88 dls/bs, % ± ±4.26 MS/OS, % ± ±19.10 MAR, mm/die ± ±0.13 BFR/BS, mm3/mm2/die ± ±0.037 Aj.AR, mm/die ± ±0.123 Mlt, days ± ±10.18 am±sd or ranges. designation of predominant hyperparathyroidism implied a metric average values (±SD) are reported in Table 1, general increase in bone turnover rate. In mixed osteodys- together with normal reference values. On average, trophy local increase in bone turnover rate coexisted with patients on dialysis have a relatively severe degree of defective mineralization. Osteomalacia was characterized by secondary hyperparathyroidism, as shown by elevated a decrease in bone turnover associated with an increase of osteoid surface and thickness. Adynamic bone disease was levels of intact PTH and high levels of several bone characterized by reduced bone turnover associated with thin markers, especially those which are normally excreted osteoid seams, bone cell paucity, and a decrease in tetracycline uptake. In the cases in which tetracycline labels were However, a wide range of values was obtained; PTH by the kidney, such as BGP, ICTP, and DPD. not available, histological diagnosis was based on the static ranged from low to markedly elevated values. histological features [20]. In our population, no cases of A correlation matrix of main humoral variables and osteomalacia were found. histomorphometric and histodynamic parameters is For an evaluation of the discriminating power of serum intact PTH and the bone markers, we divided the patients into reported in Table 2. A high level of correlation exists three different histologic groups. Patients with adynamic bone between intact PTH and AP (0.804), BALP (0.767), disease (ABD) were labeled as low turnover osteodystrophy ICTP ( 0.789), and DPD ( 0.756), respectively. High (LTO) and the remaining cases were identified as mixed values of correlation coefficients are also found between osteodystrophy (MO) or prevailing hyperparathyroidism (HP). intact PTH and several histomorphometric and histo- Statistical evaluation was carried out with linear regression dynamic parameters, such as ObS/BS, dls/bs and analysis, correlation analysis (Pearson s and Spearman s r BFR/BS (0.882, 0.753, 0.798, respectively). Total AP correlation coefficients), one way ANOVA, and Kruskall and Wallis s ANOVA [21]. Multiple comparisons of differences correlates with high degree of significance to BALP, among histologic classes was made with Bonferroni s method ICTP, as well as dls/bs and BFR/BS (0.917, 0.848, [21]. The data were also evaluated by sensitivity analysis, 0.758, and 0.900, respectively). BALP is highly correlproviding values of sensitivity and specificity per single ated with ICTP, OV/BV, dls/bs, and BFR/BS (0.938, humoral marker at a threshold value, maximizing the 0.801, 0.769, and 0.891, respectively). ICTP is highly Youden s index. In addition, data were also evaluated by a correlated with OV/BV ( 0.786), dls/bs (0.780), and standardized score approach. BFR/BS (0.807). DPD correlates satisfactorily to OS/BS (0.658), dls/bs (0.726), and BFR/BS (0.659). Results Correlation coefficients of ICTP and DPD with OcS/BS were and 0.611, respectively. The level Mean age of patients, sex distribution, mean time of correlation between BGP and TRAP is low (0.231). (months) on dialysis and humoral and histomorpho- Correlations of these variables to BFR/BS are 0.623

5 2298 G. Coen et al. Table 2. Correlation matrix of humoral and bone histomorphometric parameters Parameters Intact PTH BGP AP BALP TRAP ICTP DPD Intact PTH BGP (0.666) AP (0.885) (0.564) BALP (0.871) (0.576) (0.953) TRAP (0.352) (0.370) (0.447) (0.419) ICTP (0.742) (0.592) (0.785) (0.804) (0.423) DPD (0.730) (0.548) (0.830) (0.773) (0.506) (0.809) BV/TV (0.299) (0.384) (0.331) (0.393) (0.293) (0.414) (0.309) OV/BV (0.787) (0.691) (0.820) (0.832) (0.404) (0.751) (0.710) O.Th (0.723) (0.557) (0.793) (0.850) (0.325) (0.702) (0.675) OS/BS (0.767) (0.733) (0.788) (0.810) (0.509) (0.778) (0.697) ObS/BS (0.886) (0.686) (0.921) (0.930) (0.451) (0.787) (0.752) ES/BS (0.683) (0.515) (0.739) (0.770) (0.430) (0.661) (0.667) OcS/BS (0.724) (0.502) (0.796) (0.818) (0.453) (0.656) (0.666) sls/bs (0.818) (0.655) (0.803) (0.854) (0.307) (0.697) (0.586) dls/bs (0.756) (0.648) (0.849) (0.849) (0.603) (0.744) (0.744) MS/OS (0.588) (0.373) (0.633) (0.654) (0.370) (0.572) (0.557) MAR (0.819) (0.656) (0.841) (0.845) (0.583) (0.731) (0.726) BFR/BS (0.848) (0.710) (0.887) (0.896) (0.518) (0.771) (0.744) Aj.AR (0.732) (0.499) (0.761) (0.772) (0.501) (0.662) (0.662) Mlt ( 0.615) ( 0.412) ( 0.614) ( 0.613) ( 0.475) ( 0.527) ( 0.564) Spearman correlation coefficients in parentheses.

6 Bone markers and renal osteodystrophy 2299 and 0.731, respectively. The linear correlations among specificity, and Youden s indexes are reported. The the humoral parameters intact PTH, BGP,AP, BALP, highest level of Youden s index was given by BALP, ICTP, DPD, TRAP, and BFR/BS are reported in while DPD was at the same level as intact PTH. Figure 1. Due to the relatively low number of cases and the Classification of bone histology led to the identifica- biased balance of the data (nine LTO vs 32 MO and tion of nine cases of LTO, nine of MO and 23 patients HP), other multivariate techniques were avoided. with prevalent HP. No patients had aluminium accu- Therefore, further statistical evaluation was carried out mulation as analysed by aluminium histochemistry. employing a standardized score approach. For this Average values of the humoral and histomorphometric analysis, each variable is expressed in terms of z-score. and histodynamic parameters in the three histologic For each patient, the scores were summed and the classes, as well as the ANOVA and Bonferroni analysis, value obtained was divided by the number of humoral are reported in Table 3. For all variables, except variables. The final mean score for each patient was BV/TV, analysis of variance showed a significant found to be a good predictor of the histological class. difference among histological classes. Differences With a cut-off value of 0.6, and by classification of between humoral values of LTO and HP groups were patients having a mean score greater than 0.6 as generally significant. An analysis of discriminant power MO/HP and patients with a mean score lower than of each single humoral parameter, in identifying LTO 0.6 as LTO, a correct classification of MO/HP and vs the other two groups, MO and HP, was carried out. of LTO was possible in 93.8 and 88.9% of the cases, The results of the sensitivity analysis are shown in respectively. Predictive power was 96.8 and 80%, Table 4. The thresholds for the parameters at the level respectively for MO/HP and LTO. When the only of maximal accuracy, together with sensitivity and variables used were intact PTH and BALP, a correct Table 3. Average values (±SD) of humoral and histomorphometric parameters in the patients with LTO, MO,HP Kruskall Low turnover Mixed & Wallis osteodystrophy osteodystrophy Prevalent HP ANOVA ANOVA LTO vs MOa LTO vs HPa (LTO) (MO) (HP) P P P P Patients (n) intact PTH, pg/ml 70.97± ± ± < < <0.01 <0.01 Osteocalcin, ng/ml 43.10± ± ±98.00 < < <0.01 <0.01 AP, U/l 79.31± ± ± < <0.016 ns <0.05 BALP, mg/l 10.18± ± ± < <0.05 ns ns TRAP, U/l 4.85± ± ±3.48 <0.004 <0.01 ns <0.01 ICTP, ng/ml 44.23± ± ±97.47 < <0.017 ns <0.05 DPD, nmol/l 15.27± ± ±26.47 < < ns <0.01 BV/TV, % 18.48± ± ±6.86 ns ns ns ns OV/BV, % 2.35± ± ±8.1 < < <0.01 <0.01 O.Th, mm 9.099± ± ±6.74 < <0.012 ns <0.01 OS/BS, % 18.79± ± ±15.8 < < <0.01 <0.01 ObS/BS, % 1.12± ± ±14.14 < < <0.01 <0.01 ES/BS, % 1.985± ± ±3.74 < < <0.01 <0.01 OcS/BS, % 0.18± ± ±2.09 < < <0.01 <0.01 sls/bs, % 3.62± ± ±9.25 < < <0.05 <0.01 dls/bs, % 1.38± ± ±12.66 < < <0.01 <0.01 MS/OS, % 21.19± ± ±20.71 < < ns <0.01 MAR, mm/die 0.45± ± ±0.525 < < <0.01 <0.01 BFR/BS, mm3/mm2/die 0.016± ± ±0.412 < < <0.01 <0.01 Aj.AR, mm/die 0.095± ± ±0.519 < < <0.05 <0.01 Mlt, days ± ± ±3.24 < < <0.01 <0.01 atype one error corrected by Bonferroni s method. Number of patients for the histodynamic parameters in groups LTO, MO, and HP were 8, 8, and 21 respectively. Table 4. Sensitivity, specificity,accuracy and Youden index in the discrimination of LTO vs MO-HP. Parameters n LTO Cutting point Sensitivity Specificity Accuracy % Youden index Intact PTH, pg/ml BGP, ng/ml AP, U/l BALP, mg/l TRAP, U/l ICTP, ng/ml DPD, nmol/l

7 2300 G. Coen et al. classification of MO/HP and LTO was possible in et al. [28] and later studies have provided new data 90.6% and 88.9%, respectively. Predictive value of favouring this hypothesis at least for several target MO/HP was 96.7% and for LTO 72.7%, respectively. organs such as liver, pancreas, and kidney [26,27]. To compare the diagnostic value of AP and BALP, Certainly an additional factor of bone resistance to the same mean score method was applied to intact PTH is also bone aluminium accumulation [ 9], present PTH and AP. Correct classification of MO/HP and in several haemodialysis populations reported [29 32]. LTO was possible in 81.3 and 88.9%, respectively. Moreover, the assay used to measure intact PTH has Predictive power was 96.3 and 57.2%, respectively. been reported [33] to also detect a non (1 84)PTH Therefore AP was apparently slightly less valid than molecule, which accumulates in chronic renal failure. BALP. In general the results indicate that many humoral Whether this molecule is biologically active is not parameters carry the same quality of information known. This finding may partially explain the discrep- and the use of intact PTH and BALP in the diagnostic ancy between the results of intact PTH assays and procedure seems adequate. peripheral biological effects observed in uraemia. The results indicate that intact PTH and several Discussion other markers, AP, BALP, ICTP, and less so DPD, BGP, and TRAP, have very satisfactory correlation coefficients with most of the pertinent histomorphometric Osteodystrophy in chronic renal failure has become an and histodynamic parameters. In particular, important and widely studied disease, mainly after the excellent correlations were found between intact PTH, institution of maintenance dialysis, a treatment procedure AP, BALP, ICTP, and the parameter BFR/BS, which enabling indefinite prolongation of life and further is the most direct index of bone turnover. Also the development of bone damage. The bone lesions mainly humoral parameters, indicative of bone resorption, derive from secondary hyperparathyroidism [21]. In were strictly correlated to bone formation parameters, addition, osteomalacia in dialysis was considered to be probably due to a strict coupling between formation a separate entity and was later attributed to toxic and resorption processes in bone and to the more aluminium accumulation [2,3,23]. Therefore, osteodystrophic precise determination of bone formation histological bone patterns were classified as mild to severe parameters compared to bone resorption parameters hyperparathyroidism, mixed forms of hyperparathyroidism [17]. Serum DPD and ICTP in dialysis patients were and osteomalacia, and pure osteomalacia. significantly correlated to bone turnover parameters Another type of bone lesion was later recognized, despite accumulation in the serum due to diminished representing so called adynamic bone disease. This renal excretion. Our findings regarding DPD are in lesion is characterized by paucity of bone cells and low agreement with those of Ureña et al. [12], except for bone turnover as assessed by histodynamic parameters, the value of the correlation coefficient with BFR/BS similar to osteomalcia but without accumulation of exceeding that of intact PTH. In terms of serum ICTP, abundant osteoid tissue, i.e. with a normal osteoid our results differ from those of Ureña et al. [12], which thickness [24]. This bone lesion has emerged as a in their experience does not correlate significantly with separate and distinct type of osteopathy, unrelated to the dynamic parameters of bone turnover. Except for aluminium exposure and different from osteomalacia a considerably higher number of patients investigated [1,24]. Therefore, there are two types of low turnover with tetracycline double labeling in our study, there is bone disease, osteomalacia and ABD. In cases with no other apparent explanation for this discrepancy. In low PTH and low levels of osteocalcin and alkaline theory, serum DPD should not be expected to differ phosphatase, and the exclusion of a toxic aluminium as a marker of bone resorption from ICTP, a bone exposure by measuring serum aluminium concentra- marker considered to be a cross-linked small peptide tions, basally and after DFO administration [25], the derived from bone collagen, apparently neither metab- presence of ABD is strongly suspected. Nevertheless, olized nor reutilized in bone collagen formation. a bone biopsy is generally advocated for further therapeutic Doubts raised regarding the specificity of the antibody decisions. As previously discussed, serum intact used in the assay are not confirmed by our data [34]. PTH has always been considered the most reliable The sensitivity analysis revealed that BALP and humoral marker of bone turnover and osteodystrophic DPD, together with total AP and intact PTH, are valid pattern. Except for markedly elevated levels of intact parameters in predicting LTO. However, statistical PTH, which generally denounce a condition of hyper- analysis with z-score standardization of the humoral parathyroid-induced histological pattern, levels of the variables is more reliable in discriminating LTO from hormone in the range of 3 5 times the upper normal the other histological osteodystrophic patterns, since levels have been found to occasionally accompany the use of a single vs two or more parameters may ABD. Therefore, intact PTH is not always a good lead to misclassification, due to fluctuation of values index of the actual bone lesion [8]. The discrepancy inherent to analytical or interindividual variability. between the number of PTH dependent osteogenic Based on our results obtained with a limited number cells, such as osteoblasts and osteoclasts, and the levels of cases with LTO, multiple variable analysis may be of intact PTH, is accounted for by target organ resistance to PTH in CRF [24,26,27]. Resistance to the hormone was hypothesized many years ago by Massry limited to intact PTH and BALP. The importance of bone alkaline phosphatase in the diagnosis of LTO, compared to intact PTH and osteocalcin has been

8 Bone markers and renal osteodystrophy 2301 recently underlined by Couttenye et al. [32]. The 8. Quarles LD, Lobaugh B, Murphy G. Intact parathyroid horadvantage gained by using several markers to identify mone overestemates the presence and severity of parathyroid- mediated osseous abnormalities in uraemia. J Clin Endocrinol LTO is small, since some bone markers convey the Metab 1992; 75: same type of information. The fact that our patients 9. Fournier A, Oprisiu R, Said S, Sechet A, Ghazali A, Marie A, were not affected by cytolytic or cholestatic liver disease El Esper I, Brazier M, Achard JM, Morinière P. Invasive versus may have decreased the expected discrepancy between non invasive diagnosis of renal bone disease. Curr Opinion Nephrol Hypertens 1997; 6: the predictive values of BALP and AP. Couttenye 10. Garnero P, Delmas PD. Assessment of the serum levels of bone et al. also did not find a significant difference between alkaline phosphatases with a new immunometric assay in these markers in their study [32]. Ureña et al. [35] patients with metabolic bone disease. J Clin Endocrinol Metab found that BALP provided clearly superior results 1993; 77: than AP probably due to a less strict selection of 11. Mazzaferro S, Pasquali M, Ballanti P, Bonucci E, Costantini S, Chicca S, DeMeo S, Perruzza I, Sardella D, Taggi F, Coen G. patients without liver disease. Diagnostic value of serum peptides of collagen synthesis and Considering that our population was not seriously degradation in dialysis renal osteodystrophy. Nephrol Dial exposed to aluminium accumulation, one might Transplant 1995; 10: wonder whether the behaviour of intact PTH and the 12. Ureña P, Ferreira A, Kung VT, Morieux C, Simon P, Ang KS, Souberbielle JC, Segre GV, Drueke TB, DeVernejoul MC. Serum other bone markers might be different from dialysis pyridinoline as a specific marker of collagen breakdown and populations with bone aluminium accumulation. Our bone metabolism in hemodialysis patients. J Bone Miner Res cut-off values for intact PTH are lower than in other 1995; 10: reports [29] and could be due to differences in alumidependent 13. Price PA, Nishimoto SR. Radioimmunoassay for the vitamin K protein of bone disease and its discovery in plasma. nium exposure. Proc Natl Acad Sci USA 1980; 77: In conclusion a bone biopsy should be performed in 14. Bianco P, Ponzi A, Bonucci E. Basic and special stains for cases with borderline PTH and serum marker concen- plastic sections in bone marrow histopathology, with special trations. Performance of bone biopsy remains obligat- reference to May-Grunwald-Giemsa and enzyme histochemistry. ory in cases of suspected aluminium toxicity, a Basic Appl Histochem 1984; 28: condition preventing parathyroidectomy and requiring 15. Maloney NAQ, Ott SM, Alfrey AC, Miller NL, Coburn JW, Sherrard DJ. Histological quantification of aluminium in iliac DFO detoxication. Even though serum aluminium and bone from patients with renal failure. J Lab Clin Med 1982; DFO test may be normal in cases which are not 99: currently exposed to aluminium ingestion, a bone 16. Parfitt AM, Drezner MK, Glorieux FH, Kanis JA, Malluche biopsy should also be performed in cases with a former HH, Meunier PJ, Ott SM, Recker RP. Bone histomorphometry: standardization of nomenclature, symbols and units. Bone Miner history of prolonged and severe aluminium exposure 1987; 2: [24,36]. 17. Bonucci E, Ballanti P, DellaRocca C, Milani S, LoCascio V, Imbimbo B. Technical variability of bone histomorphometric Acknowledgements. The study has been supported by funds from measurements. Bone Miner 1990; 11: Ministero della Universita e Ricerca Scientifica and La Sapienza 18. Ballanti P, Bonucci E, DellaRocca C, Milani S, LoCascio V, University of Rome. 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